The (+) chain of satellite RNA of tobacco ringspot virus and the (+) chain and (-) chain of avocado sunblotch viroid are cleaved by their own catalytic activity in the presence of Mg.sup.2+ (Science 231, 1577-1580 (1986)). The RNA structure necessary for this cleavage activity has been determined and named as Hammerhead ribozyme (Nucleic Acids Res. 14, 3627-3640 (1986)). The nucleotide sequences in the vicinity of the cleavage sites of these RNA possesses common sequences, and the secondary structure of these RNA was predicted from this common sequences. Uhlenbeck designed a short chain RNA fragment of 19 mer based on these common sequences, and indicated that said fragment catalytically cleaves RNA of 24 mer (Nature, 328, 596-600 (1987)).
In addition, besides viroid and vital satellite RNA, the transcript of newt satellite DNA is also reported to have ribozyme nucleotide sequences (Cell, 48, 535-543, (1987)).
The inventors of the present invention chemically synthesized two types of 21 mer RNA having nucleotide sequences in the vicinity of the cleavage site of this newt satellite DNA transcript. When one of the RNA was added to the other, a cleavage reaction was found to occur at the same site as that in nature (FEBS Lett., 228, 228-230, (1988)). In addition, based on this result, the inventors of the present invention found a process for cleaving other RNA or polyribonucleotide molecules using ribozyme (Nucleic Acids Res., 17, 7059-7071, (1989)).
On the other hand, a cleavage reaction was also caused on the (-) chain of the satellite RNA of tobacco ringspot virus, and that cleavage has been determined to occur at a specific site (Nature 323, 349-353 (1986)). In addition, the minimum region of RNA necessary for this cleavage has also been recently clarified (Biochemistry, 28, 4929-4933 (1989)). RNA having this catalytic activity is composed of 50 nucleotides, and a model having a hairpin loop structure within this RNA has been advocated. This RNA has been given the name hairpin ribozyme. The present inventors and other research groups converted the nucleotides of this hairpin ribozyme to other nucleotides, and used those results to identify several nucleotides that are important in the cleavage reaction (Nature 354, 320-322 (1991), Nucleic Acids Res. 19, 6833-6838 (1991)). Further, the group of Burke et al. has recently identified the base sequence which is important in the cleavage reaction and ligation reaction of hairpin ribozyme by in vitro selection method using DNA having random variation and PCR (polymerase chain reaction) (Gene & Development 6, 129-134 (1992)). The present inventors have determined that catalytic reaction proceeds even for RNA deleting the hairpin loop portion (Nucleic Acids Res. 19, 6833-6838 (1991)).
In addition, progress in RNA synthesis techniques in recent years has made it possible to obtain RNA in large volume, and thus, researches on higher-order structures of RNA and its physicochemical properties are increasingly made. Bacteriophage T.sub.4 mRNA and E. coli 16S ribosomal RNA contain 5'CUUCGG3' sequences in high frequency, and it has been determined that the hairpin loop structure formed by this sequence is thermodynamically stable (Pro. Natl. Acad. Sci. USA, 85, 1364-1368, (1988): Nature, 346, 680-682, (1990)).
The present inventors prepared a polyribonucleotide wherein the nucleotide sequence of a hairpin loop which exists at one location within the ribozyme is the above-mentioned thermodynamically stable 5'CUUCGG3' sequence, and they found that this polyribonucleotide possesses high ribozyme cleavage activity and accomplished the present invention. As a result of having a thermodynamically stable hairpin loop structure, the ribozyme in the present invention is expected to efficiently cleave target polyribonucleotides in the living body.